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CI Research Report: 2006

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

I was able to carry out 140 experiments for this year. The experiments were devoted to several research projects.

1. I continued to study the possibility of preservation of patients’ brains during 12-24 hour transportation from regions which are far from CI.

1.1 I finished the study of warm and cold ischemia of rat brains. The results were published in the Immortalist magazine (2006, vol. 38, No. 1-2 and 5-6). The main conclusion is that no present organ preservation solutions and additives were useful for long-term cold storage of the brain. Cryonics is very different from today cryobiology and medicine because it relies on future perfect technology, but today imperfect cryobiology and medicine can use only natural, spontaneous resources of biological systems to recover them from ischemic damage.

1.2 I continued to study Dr. Suda’s experiments using whole rat brains and the K/Na ratio assay. The brains were perfused with glycerol solutions (5, 10, and 15% v/v) at 0ºC, slowly cooled to −20ºC, kept at this temperature for 12-24 hours, and washed out from glycerol. Survival of the rat cerebral tissues was evaluated by the hippocampal slice method I usually used. The tissues were dead.

1.3 I continued to study Dr. Seki’s experiments using rat cerebral tissues. Dr. Seki dehydrated rat hearts in a certain extent and kept them in a non-aqueous medium. He briefly reported in the Cryobiology journal in 1999 that he was able to preserve and resuscitate the rat hearts after 10-26 day cold storage! However, there was none of his articles or patent applications on this subject for 2000 – 2006.

I tried to use 2.5-20% glucose solutions in order to dehydrate rat brains in various extents for an improvement of cold storage of the brains. However, that osmotic dehydration was harmful for the rat brains.

I also tried to use Dr. Seki procedure for rat hippocampuses. The hippocampuses were stored in inert fluid with silica gel at 2-4ºC for 24 hours. There was no positive effect.

Rat hippocampus were dried with silica gel in 20 ml vials without inert fluid at 2-4ºC for various time and subsequent cold storage of the hippocampuses for 24 hours. The experiments were performed to find the influence of various degrees of dehydration of rat hippocampuses by drying on slice survival. There was no positive effect again.

I would like to publish the results of my experiments on Dr. Suda’s and Dr. Seki’s subjects in the Immortalist magazine.

2. I continued to study the quality of vitrification mixture (VM) perfusion for sheep brains that had 24 hour cold ischemia.

It is an important research for CI because most CI patients had long term (12-24 hour) cold ischemia. My experiments showed that the sheep heads were successfully perfused with VM-1 solutions even after their storage at 2-4ºC for 24 hours! The sheep brains had the stable vitrification. It is a very important positive result for us. I also did not observe any breakage of the cerebral blood vessels.

3. I studied some problems of VM perfusion of the body. Saturation of the human body with any vitrification mixtures requires a very large amount of VMs to get its uniform and stable vitrification.

A main problem was an accumulation of a large amount of perfusates in the alimentary tract and abdominal cavity. An exact cause of this phenomenon is not known. Most likely, there was a leakage of blood capillaries in those regions. For now, it is impossible to saturate a patient’s body with vitrification mixtures to get its uniform and stable vitrification.

In the past CI had a little swelling of patients’ abdomens because CI used a very small amount of glycerol solutions for body perfusion. Sometimes CI had a bigger swelling even with the small amount of glycerol solutions because those CI patients had a cancer of inner organs.

I elaborated a method of perfusion of a patient’s body with ethylene glycol (EG) solutions for freezing procedure but not for vitrification one. Concentrated EG solutions are much less viscous than glycerol ones. EG better penetrates in tissues than glycerol.

4. I have written a patent application for the CI method of cryopresevation of cerebral tissues by vitrification.

I needed to perform several additional experiments for the application. Cryonics Institute's vitrification mixture (CI-VM-1) is very simple and cheap. This is one of most important advantages of CI-VM-1 in comparison with other known VMs.

5. I continued to study the possibility of an improvement of the present CI vitrification method for potential CI patients without long term ischemia.

12-24 hour cold ischemia decreased survival of cerebral tissues to 40% of the control. We should propose CI members an improved CI vitrification method with maximally possible cryopreservation of cerebral tissues. For this, first of all, potential CI patients should avoid the long term ischemia.

I think usual compounds that were used against short term ischemia (i. e. anti-oxidants) cannot improve long term (12-36 hours) cold storage of the brain. I decided to test some of unusual compounds. But I have just a little hope to improve the results of the long term ischemia.

One of harmful factors is excessive dehydration of the brain during VM perfusion. The cause of this problem is a very low permeability of the blood-brain barrier (BBB) for cryoprotective agents. As I reported previously, I was able to saturate rat and sheep brains with VM-1 completely without dehydration using Substance X to open BBB. However, I need to continue the study of the influence of brain dehydration on cerebral tissue survival in order to find an optimal procedure for opening BBB. The procedure could be material for a new patent application.

Saturation of the human brain with 70% VM-1 at -20ºC instead of 0ºC could significantly increase cerebral tissue survival. The main problem is very high viscosity of 70% VM-1 at −20ºC, although CI-VM-1 is less viscous than other VMs. I need to perform more experiments to try overcoming the problem.

I also performed experiments studying the possibility of faster cooling a patient’s head through its blood vessels using inert fluids after saturation of the head with VMs. The models for the human head were rat and sheep heads.

Ben Best proposed me to use trehalose and sucrose in order to try decreasing CPA concentrations in VM-1. I was and now I am against decreasing CPA concentrations in VM-1 because this will decrease the stability of brain vitirfication and so can result in devitrification and ice formation. My experiments with trehalose and sucrose showed the decreasing of the stability of cerebral slice vitirfication. Ice crystallization is a very powerful process and so we should not decrease VM-1 concentration. I propose another way to increase cerebral tissue survival, namely to increase the resistance of cerebral tissues to toxic effects of VM-1.